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J. Biol. Chem., Vol. 280, Issue 27, 25436-25449, July 8, 2005

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The Broadly Selective Human Na⁺/Nucleoside Cotransporter (hCNT3) Exhibits Novel Cation-coupled Nucleoside Transport Characteristics^*

Kyla M. Smith, Melissa D. Slugoski¶, Shaun K. Loewen¶, Amy M. L. Ng, Sylvia Y. M. Yao, Xing-Zhen Chen, Edward Karpinski, Carol E. Cass||**, Stephen A. Baldwin, and James D. Young¶¶

From the Membrane Protein Research Group, Departments of Physiology and^|| Oncology, University of Alberta, and the^** Cross Cancer Institute, Edmonton, Alberta T6G 2H7, Canada and the School of Biochemistry and Microbiology, University of Leeds, Leeds LS2 9JT, United Kingdom

Received for publication, August 17, 2004 , and in revised form, March 24, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The concentrative nucleoside transporter (CNT) protein family in humans is represented by three members, hCNT1, hCNT2, and hCNT3. hCNT3, a Na⁺/nucleoside symporter, transports a broad range of physiological purine and pyrimidine nucleosides as well as anticancer and antiviral nucleoside drugs, and belongs to a different CNT subfamily than hCNT1/2. H⁺-dependent Escherichia coli NupC and Candida albicans CaCNT are also CNT family members. The present study utilized heterologous expression in Xenopus oocytes to investigate the specificity, mechanism, energetics, and structural basis of hCNT3 cation coupling. hCNT3 exhibited uniquely broad cation interactions with Na⁺, H⁺, and Li⁺ not shared by Na⁺-coupled hCNT1/2 or H⁺-coupled NupC/CaCNT. Na⁺ and H⁺ activated hCNT3 through mechanisms to increase nucleoside apparent binding affinity. Direct and indirect methods demonstrated cation/nucleoside coupling stoichiometries of 2:1 in the presence of Na⁺ and both Na⁺ plus H⁺, but only 1:1 in the presence of H⁺ alone, suggesting that hCNT3 possesses two Na⁺-binding sites, only one of which is shared by H⁺. The H⁺-coupled hCNT3 did not transport guanosine or 3'-azido-3'-deoxythymidine and 2',3'-dideoxycytidine, demonstrating that Na⁺- and H⁺-bound versions of hCNT3 have significantly different conformations of the nucleoside binding pocket and/or translocation channel. Chimeric studies between hCNT1 and hCNT3 located hCNT3-specific cation interactions to the C-terminal half of hCNT3, setting the stage for site-directed mutagenesis experiments to identify the residues involved.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Physiological nucleosides and synthetic nucleoside analogs have important biochemical, physiological, and pharmacological activities in humans. Adenosine, for example, has purino-receptor-mediated functions in processes such as modulation of immune responses, platelet aggregation, renal function, and coronary vasodilation (1, 2). Nucleosides also provide salvage precursors for nucleic acid biosynthesis, and nucleoside drugs are commonly used in the therapy of cancer and viral infections (3, 4). Most nucleosides, including those with antineoplastic and/or antiviral activities, are hydrophilic and require specialized plasma membrane nucleoside transporter (NT)¹ proteins for their uptake into or release from cells (5–7).

Multiple transport systems for nucleosides have been observed in human and other mammalian cells and tissues (7–9). The concentrative systems (cit, cif, and cib)² are inwardly directed Na⁺-dependent processes present primarily in intestinal and renal epithelia and other specialized cells (7–9). The equilibrative systems (es and ei) mediate bidirectional downhill transport of nucleosides, have generally lower substrate affinities than the concentrative systems, and occur in most, possibly all, cell types (7–9). Systems cit and cif transport adenosine and uridine, but are otherwise pyrimidine and purine nucleoside-selective, respectively, whereas systems cib, es, and ei transport both pyrimidine and purine nucleosides. System es is inhibited by nanomolar concentrations of nitrobenzylthioinosine (NBMPR), whereas system ei also transports nucleobases (7–10).

Molecular cloning studies have identified the human and rodent integral membrane proteins responsible for each of these nucleoside transport activities (11–18). They belong to two previously unrecognized and structurally unrelated protein families (CNT and ENT), and their relationship to the processes defined by functional studies is: CNT1 (cit), CNT2 (cif), CNT3 (cib), ENT1 (es), and ENT2 (ei) (11–18). In addition to ENT1 and ENT2, the ENT protein family also contains three further human and rodent members (ENT3, ENT4, and CLN3) (19–23). Human and other eukaryote CNTs have 13 predicted transmembrane helices (TMs), with an intracellular N terminus and an extracellular C terminus (24). NupC, an H⁺-coupled CNT from Escherichia coli, has a similar predicted topology, but lacks TMs 1–3 (25, 26). Other CNT family members that have been functionally characterized include H⁺-coupled CeCNT3 and CaCNT from Caenorhabditis elegans (27) and Candida albicans (28), respectively, and Na⁺-coupled hfCNT from the Pacific hagfish (Eptatretus stouti) (29, 30).

Human and mouse CNT3 (hCNT3 and mCNT3) are the most recent mammalian CNT family members to be identified (18) and, together with cib-type hagfish CNT (hfCNT) (30), form a phylogenetic CNT subfamily separate from the mammalian CNT1/2 subfamily (18). In addition to differences in substrate selectivities, members of the two subfamilies also differ in the stoichiometry of Na⁺/nucleoside coupling. Contrary to a recent report in this journal (31), for example, we have shown that hCNT1 has a Na⁺/nucleoside coupling ratio of 1:1 (32), whereas the coupling ratio of hfCNT is 2:1 (30). Here, we extend these investigations and present a mechanistic and chimeric analysis of cation coupling in hCNT3. The results validate our previously reported differences in cation coupling between the CNT3/hfCNT and CNT1/2 subfamilies and reveal additional novel features of CNT3-cation interactions.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
hCNT3 cDNA—cDNA encoding hCNT3 (GenBank^TM accession number AF305210 [GenBank] ) in the enhanced Xenopus plasmid expression vector pGEM-HE (33) with flanking 5'- and 3'-untranslated regions from the Xenopus -globin gene was obtained as previously described (18).

In Vitro Transcription and Expression in Xenopus Oocytes—hCNT3 plasmid DNA was linearized with NheI and transcribed with T7 polymerase using the mMESSAGE mMACHINE^TM (Ambion, Austin, TX) transcription system. The remaining template was removed by digestion with RNase-free DNase1. Stages V–VI oocytes from Xenopus laevis were treated with collagenase (2 mg/ml) for 2 h, and remaining follicular layers were removed by phosphate treatment (100 mM K₂PO₄) and manual defolliculation (11). Twenty-four hours after defolliculation, oocytes were injected with either 10 nl of water containing 10 ng of RNA transcript encoding hCNT3 or 10 nl of water alone. Injected oocytes were then incubated for either 4 days (radioisotope flux studies) or 4–7 days (electrophysiology) at 18 °C in modified Barth's solution (changed daily) (88 mM NaCl, 1 mM KCl, 0.33 mM Ca(NO₃)₂, 0.41 mM CaCl₂, 0.82 mM MgSO₄, 2.4 mM NaHCO₃, 10 mM Hepes, 2.5 mM sodium pyruvate, 0.05 mg/ml penicillin, and 0.1 mg/ml gentamycin sulfate, pH 7.5).

Transport Media—Unless otherwise stated, electrophysiological and radioisotope flux studies used Na⁺-containing transport medium composed of 100 mM NaCl, 2 mM KCl, 1 mM CaCl₂, 1mM MgCl₂, and 10 mM Hepes (for pH values 6.5) or 10 mM MES (for pH values 6.5). In experiments examining the Na⁺ dependence of transport (i.e. where the indicated Na⁺ concentration is <100 mM), Na⁺ in the transport medium was replaced by equimolar choline chloride (ChCl) to maintain isomolarity. Proton dependence was tested in Na⁺-free choline-containing transport medium (100 mM ChCl) at pH values ranging from 4.5 to 8.5. Experiments testing Li⁺ coupling of transport were performed in medium at pH 8.5 containing Li⁺ (100 mM LiCl) in place of Na⁺.

Electrophysiological Studies—Nucleoside-evoked membrane currents were measured in hCNT3-producing oocytes at room temperature (20 °C) using a GeneClamp 500B oocyte clamp (Axon Instruments Inc., Foster City, CA) in the two-electrode, voltage clamp mode. The GeneClamp 500B was interfaced to an IBM-compatible PC via a Digidata 1200A/D converter and controlled by pCLAMP software (version 8.0, Axon Instruments Inc.). The microelectrodes were filled with 3 M KCl and had resistances that ranged from 0.5 to 2.5 megohms. Oocytes were penetrated with the microelectrodes, and their membrane potentials were monitored for periods of 10–15 min. Oocytes were discarded when membrane potentials were unstable, or more positive than –30 mV. Unless otherwise indicated, the oocyte membrane potential was clamped at a holding potential (V_h) of –50 mV, and nucleoside was added in the appropriate transport medium at a concentration of 100 µM. Current signals were filtered at 20 Hz (four-pole Bessel filter) and sampled at a sampling interval of 20 ms. For data presentation, the signals were further filtered at 0.5 Hz by the pCLAMP program suite.

In protonophore studies, carbonyl cyanide m-chlorophenylhydrazone (CCCP) (100 µM) was preincubated with oocytes for 15 min prior to measuring uridine-evoked currents (100 µM) in Na⁺-free medium (100 mM ChCl, pH 5.5). Stock solutions of CCCP were dissolved in Me₂SO. Control experiments confirmed that oocytes were unaffected by Me₂SO at its final concentration of 0.5% (w/v).

View larger version (19K): [in this window] [in a new window]   FIG. 1. Effects of Na+, H+, and Li+ on the transport activities of oocytes expressing recombinant hCNT3. Radiolabeled fluxes of uridine (20 µM, 20 °C, 1-min flux) in hCNT3-producing oocytes were measured in transport media containing Na+ (black bar; 100 mM NaCl, pH 7.5), choline (open bars; 100 mM ChCl, pH 5.5–8.5), or Li+ (gray bar; 100 mM LiCl, pH 8.5). Values were corrected for basal non-mediated uptake in control water-injected oocytes and are means ± S.E. of 10–12 oocytes. The experiment was performed on a single batch of oocytes used on the same day.

Data from individual electrophysiology experiments are presented as nucleoside-evoked currents from single representative cells or as mean values (±S.E.) from four or more oocytes from the same batch of oocytes used on the same day. Each experiment was repeated at least twice on oocytes from different frogs. No nucleoside-evoked currents were detected in oocytes injected with water alone, demonstrating that currents in hCNT3-producing oocytes were transporter-specific.

Radioisotope Flux Studies—Radioisotope transport assays were performed as described previously (11, 18) on groups of 10–12 oocytes at 20 °C using ¹⁴C-labeled nucleosides (1 µCi/ml) in 200 µl of the appropriate transport medium. Unless otherwise stated, nucleoside uptake was determined at a concentration of 20 µM. Following incubation, seven rapid washes in ice-cold choline chloride transport medium (pH 7.5) removed extracellular label, and individual oocytes were dissolved in 1% (w/v) SDS for quantitation of cell-associated radioactivity by liquid scintillation counting (LS 6000 IC, Beckman, Fullerton, CA). Uptake values represent initial rates of transport (1-min flux) and are presented as means ± S.E. of 10–12 oocytes from representative experiments. Individual experiments were performed on cells from single batches of oocytes used on the same day. Transporter-mediated fluxes were calculated as uptake in RNA transcript-injected oocytes minus uptake in control water-injected oocytes. Each experiment was repeated at least twice using oocytes from different frogs.

Kinetic Parameters—Kinetic parameters calculated from electrophysiological and radioisotope flux experiments were determined by least squares fits to the Michaelis-Menten and Hill equations using Sigmaplot 2000 software (Jandel Scientific Software, San Rafael, CA). Those from electrophysiological experiments were determined from fits to data from individual oocytes normalized to the I_max value obtained for that oocyte, and are presented as values (±S.E.) for single representative oocytes, or as means (±S.E.) of four or more cells. Those from radioisotope experiments were derived from curve fits to averaged mediated data from 10–12 oocytes, and are presented as means (±S.E.).

Charge-to-Nucleoside Stoichiometry—Na⁺/nucleoside and H⁺/nucleoside coupling ratios for hCNT3 were determined by simultaneously measuring Na⁺ or H⁺ currents and [¹⁴C]uridine (200 µM, 1 µCi/ml) uptake under voltage clamp conditions. Individual hCNT3-producing oocytes were placed in a perfusion chamber and voltage clamped at V_h of –30, –50, or –90 mV in the appropriate nucleoside-free medium for a 10-min period to monitor baseline currents. When the baseline was stable, the nucleoside-free medium was exchanged with medium of the same composition containing radiolabeled uridine. Current was measured for 3 min, and uptake of uridine was terminated by washing the oocyte with nucleoside-free medium until the current returned to baseline. The oocyte was then transferred to a scintillation vial and solubilized with 1% (w/v) SDS for quantitation of oocyte-associated radioactivity. Nucleoside-induced current was obtained as the difference between baseline current and the inward uridine current. The total charge translocated into the oocyte during the uptake period was calculated from the current-time integral and correlated with the measured radiolabeled flux for each oocyte to determine the charge/uptake ratio. Basal [¹⁴C]uridine uptake was determined in control water-injected oocytes (from the same donor frog) under equivalent conditions and used to correct for endogenous non-mediated uridine uptake over the same incubation period. Coupling ratios (±S.E.) were calculated from slopes of least-squares fits of uridine-dependent charge versus uridine accumulation for eight or more oocytes.

View larger version (13K): [in this window] [in a new window]   FIG. 2. Cation/nucleoside currents. A, representative cation/nucleoside current traces in a single hCNT3-producing oocyte clamped at –50 mV in transport medium containing either Na+ (100 mM NaCl, pH 8.5), choline (100 mM ChCl, pH 5.5), or Li+ (100 mM LiCl, pH 8.5). The bar denotes addition of uridine (100 µM) to the bath. No currents were observed in control water-injected oocytes. B, averaged inward currents in hCNT3-producing oocytes perfused sequentially with 100 µM uridine in Na+-containing transport medium (black bar; 100 mM NaCl, pH 8.5), Na+-free transport medium (open bar; 100 mM ChCl, pH 5.5), and Na+-free transport medium containing 100 µM CCCP (gray bar; 100 mM ChCl, pH 5.5). Currents are means ± S.E. of five different oocytes from the same batch of cells used on the same day.

Construction of Chimeric hCNT3 and hCNT1 Transporters—hCNT1 cDNA (GenBank^TM accession number U62968 [GenBank] ) (14) was subcloned into the vector pGEM-HE prior to chimera construction with hCNT3 (already in pGEM-HE) to enhance expression in Xenopus oocytes. Two sets of overlap primers were designed at a splice site between Lys³¹⁴ and Val³¹⁵ of hCNT3 and the corresponding residues in hCNT1 (Lys²⁹³ and Ile²⁹⁴) in the putative loop linking TM 6 and TM 7 (arrow in Fig. 11A) to create reciprocal 50:50 chimeras by a two-step overlap extension PCR method (30, 34). Chimeric constructs containing the restriction site KpnI downstream of the M13 forward primer and the restriction site SphI upstream of the M13 reverse primer were subcloned into the respective restriction sites of the pGEM-HE vector. The chimeras were sequenced in both directions to verify the splice sites and ensure that no mutations had been introduced.

Chemicals—Nucleosides and CCCP were purchased from Sigma. The ¹⁴C-labeled nucleosides were purchased from Moravek Biochemicals (Brea, CA) or Amersham Biosciences. ²²Na⁺ was obtained from Amersham Biosciences.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cation Dependence of hCNT3—We have previously used heterologous expression in Xenopus oocytes in combination with radioisotope flux assays and the two-microelectrode voltage clamp to demonstrate that recombinant hCNT3 functions as a broad specificity cib-type electrogenic Na⁺/nucleoside symporter (18). The experiment of Fig. 1 extended these findings and demonstrated the effect of an imposed H⁺ gradient on the initial rate of hCNT3-mediated uptake (influx) of [¹⁴C]uridine (20 µM) measured at extracellular pH values ranging from 5.5 to 8.5 under Na⁺-free conditions. Choline (Ch⁺) was substituted for Na⁺, and values were corrected for basal non-mediated uptake in control water-injected oocytes (<0.03 pmol/oocyte·min^–1 under all conditions tested). A marked pH dependence of uridine influx was apparent. In 100 mM ChCl at pH 5.5, for example, hCNT3-mediated uridine influx was 26- fold higher than at pH 8.5, and was 60% of that obtained in the presence of 100 mM NaCl at pH 7.5. In the absence of either H⁺ or Na⁺, hCNT3 also exhibited Li⁺-dependent uridine influx (100 mM LiCl, pH 8.5, versus 100 mM ChCl, pH 8.5). In marked contrast, hCNT1 and hCNT2, the two other human CNT isoforms, showed no pH-dependent uridine uptake and exhibited very small Li⁺-mediated uridine influx (2% of the corresponding Na⁺-mediated uridine flux; data not shown). As illustrated in Fig. 2, the novel H⁺ and Li⁺ dependence of hCNT3 were further investigated by electrophysiology. External application of 100 µM uridine to a representative hCNT3-producing oocyte clamped at –50 mV under Na⁺-free conditions elicited inward H⁺ and Li⁺ currents that returned to baseline upon removal of substrate (Fig. 2A). As demonstrated by the mean current data in Fig. 2B, H⁺ currents were markedly inhibited by pre-treatment of oocytes with the protonophore CCCP. No currents were observed in control water-injected oocytes (data not shown). Thus, in addition to being Na⁺-dependent, hCNT3 functioned as an electrogenic H⁺/nucleoside and Li⁺/nucleoside symporter. These findings were also confirmed in parallel studies of mCNT3 (data not shown). Subsequent experiments focused on the mechanism(s) of Na⁺ and H⁺ coupling of hCNT3.

View larger version (33K): [in this window] [in a new window]   FIG. 3. Substrate selectivity and pH dependence of nucleoside transport by recombinant hCNT3. Oocytes were injected with either water alone or water containing hCNT3 RNA transcripts. A, averaged nucleoside-induced inward currents measured by perfusing hCNT3-producing oocytes with 100 µM pyrimidine (uridine, thymidine, and cytidine) or purine (adenosine, inosine, and guanosine) nucleosides in Na+-containing transport medium (100 mM NaCl, pH 7.5). Currents are means ± S.E. of 5–6 different oocytes from the same batch of cells used on the same day. B, mean nucleoside-induced inward currents measured by perfusing hCNT3-producing oocytes with 100 µM pyrimidine and purine nucleosides in Na+-free transport medium (100 mM ChCl, pH 5.5 and 8.5). Currents are means ± S.E. of 5–6 oocytes from the same batch of cells used on the same day. C, mediated radiolabeled fluxes of pyrimidine and purine nucleosides (20 µM, 20 °C, 1-min flux) in oocytes injected with hCNT3 RNA transcripts measured in Na+-free transport media (100 mM ChCl, pH 5.5 and 8.5). Mediated transport was calculated as uptake in RNA-injected oocytes minus uptake in control water-injected oocytes. Each value represents the mean ± S.E. of 10–12 oocytes obtained from the same batch of cells and used on the same day. D, mean nucleoside-induced inward currents measured by perfusing hCNT3-producing oocytes with cytidine, inosine, or guanosine (100 µM and 1 mM) in Na+-free transport medium (100 mM ChCl, pH 5.5 and 8.5). Currents are means ± S.E. of 5–6 oocytes from the same batch of cells used on the same day. In A, B, and D, no currents were observed in control water-injected oocytes.

View larger version (16K): [in this window] [in a new window]   FIG. 4. Nucleoside drug-induced currents in hCNT3-producing Xenopus oocytes. A, representative inward currents induced by gemcitabine (100 µM), AZT (1 mM), or ddC (1 mM) in an hCNT3-producing oocyte in transport medium containing either Na+ (100 mM NaCl, pH 7.5) or choline (100 mM ChCl, pH 5.5). Uridine-evoked currents in the same hCNT3-producing oocyte were 210 and 90 nA in Na+-containing and choline-containing transport media, respectively. B, a comparison of hCNT3-mediated inward currents following sequential addition of uridine (100 µM), gemcitabine (100 µM), AZT (1 mM), or ddC (1 mM) to transport media containing either Na+ (100 mM NaCl, pH 7.5) or choline (100 mM ChCl, pH 5.5). Currents are means ± S.E. for three different oocytes from the same batch of cells used on the same day. No currents were observed in control water-injected oocytes.

View larger version (19K): [in this window] [in a new window]   FIG. 5. Voltage dependence of hCNT3-mediated currents. A, time course of transmembrane currents recorded from a representative hCNT3-producing oocyte in the presence of 100 µM external uridine (100 mM NaCl, pH 8.5) in response to 300-ms voltage pulses from a holding potential of –50 mV (left trace). Currents are shown at four different test potentials only (Vt = +50, –10, –60, and –110 mV). The capacitive transients have been truncated to clearly demonstrate the steady-state currents. The right trace shows corresponding currents from the same oocyte recorded in the same transport medium in the absence of uridine. The records are offset to zero. B, the current-voltage (I-V) curves for hCNT3 were generated from the difference between steady-state currents recorded in the presence and absence of 100 µM uridine in Na+-containing (open circles; 100 mM NaCl, pH 8.5) or choline-containing (solid circles; 100 mM ChCl, pH 5.5) transport media upon voltage pulses from Vh of –50 mV to final potentials ranging between +60 and –110 mV, in 10-mV steps. The data are from the same oocyte as in A. No uridine-induced currents were observed in control water-injected oocytes.

Voltage Dependence of hCNT3 Transport Currents—Fig. 5A shows representative current traces in an hCNT3-producing oocyte in Na⁺-containing medium (pH 8.5) before and after perfusion with 100 µM uridine in an experiment undertaken to examine the effects of membrane potential on uridine-induced steady-state currents. Currents evoked by uridine at potentials between +60 and –110 mV were voltage-dependent and increased as the membrane potential became more negative (Fig. 5B). Uridine-induced Na⁺ currents approached zero but did not reverse polarity at potentials up to +60 mV. Measured in the same oocyte, uridine-induced H⁺ currents (ChCl, pH 5.5) were approximately half those in Na⁺-containing medium and exhibited a similar voltage dependence. In other experiments performed over a wider V_h range, uridine-induced Na⁺ and H⁺ currents did not saturate at negative potentials up to –150 mV (data not shown).

Voltage Dependence of hCNT3 Transport Kinetics—The effects of membrane potential on hCNT3 transport kinetics in the presence of external Na⁺ were examined in detail by measuring the apparent affinities for uridine (K_m) and Na⁺ (K₅₀) and the uridine-evoked maximum current (I_max) at four different holding potentials (V_h =–10, –30, –50, and –70 mV) (Fig. 6). K_m was determined in transport medium containing either 10 or 100 mM NaCl (pH 8.5), and mean values plotted as a function of V_h are summarized in Fig. 6C. As representative examples of the kinetic data used to generate K_m values, Fig. 6, A and B, show uridine concentration dependence curves recorded from individual oocytes at a membrane potential of –10 mV and external Na⁺ concentrations of 10 and 100 mM, respectively. K₅₀ was determined at an external uridine concentration of 100 µM (pH 8.5), and mean values plotted as a function of V_h summarized in Fig. 6E. The representative single oocyte Na⁺-activation curve shown in Fig. 6D was measured at a holding potential of –10 mV. I_max values were determined independently at a saturating external uridine concentration of 100 µM and a Na⁺ concentration of 100 mM (pH 8.5) (Fig. 6F).

As demonstrated in Fig. 6C, K_m was unaffected by membrane potential at 100 mM external Na⁺, but was voltage-dependent at 10 mM Na⁺, decreasing from 24 to 7.3 µM as the membrane potential was made more negative. At high negative potentials, the K_m value at 10 mM NaCl approached that observed at 100 mM external Na⁺, suggesting that the effect of membrane potential on K_m was predominantly the result of voltage dependence of Na⁺ binding (35, 36). Consistent with this, we found that the Na⁺ K₅₀ value decreased from 5.3 mM at –10 mV to 2.1 mM at –70 mV (Fig. 6E). As shown in Fig. 6F, I_max also increased as the membrane potential was made more negative. At a holding potential of –10 mV (100 mM NaCl), the mean uridine-evoked inward current was 37 nA. This increased to 97 nA at –70 mV, a trend that was similar to the I-V relationship shown in Fig. 5B. Calculated over the same limited V_h range (–10 to –70 mV), I-V curves for five oocytes gave an e-fold change (±S.E.) in current per 77 ± 4 mV, compared with 67 ± 11 mV for the data in Fig. 6F. I_max reflects movement of the loaded and empty carrier (36). Similar to the Na⁺/glucose cotransporter SGLT1, therefore, membrane potential influenced both ion binding and carrier translocation (35, 36).

View larger version (24K): [in this window] [in a new window]   FIG. 6. Voltage dependence of the transport kinetics of hCNT3. A, uridine concentration-response curve measured in a single representative hCNT3-producing oocyte at an external Na+ concentration of 10 mM (pH 8.5) and a membrane potential of –10 mV. Currents at each uridine concentration were normalized to the fitted Imax value for that oocyte (±S.E.) of 52 ± 1 nA. The Km value was 32 ± 3 µM. B, recordings from a single representative hCNT3-producing oocyte demonstrating a corresponding experiment at the same membrane potential (–10 mV) in the presence of 100 mM external Na+ (pH 8.5). Currents were normalized to the fitted Imax of 55 ± 3 nA. The Km was 7.6 ± 1.1 µM. C, the effect of membrane potential on uridine Km was determined at external Na+ concentrations of 10 mM (open circles) and 100 mM (solid circles) (pH 8.5) and holding potentials of –10, –30, –50, and –70 mV. Km values at each membrane potential were obtained from fits to data from individual oocytes normalized to the Imax value obtained for that cell and are presented as means ± S.E. of 4–8 oocytes. D, Na+ concentration-response curve (pH 8.5) measured in a single representative hCNT3-producing oocyte at a membrane potential of –10 mV (100 µM uridine). Currents at each Na+ concentration were normalized to the fitted Imax value of 43 ± 2 nA. The Na+ K50 was 5.5 ± 0.5 mM. E, the effect of membrane potential on Na+ K50 was determined at holding potentials of –10, –30, –50, and –70 mV (100 µM uridine, pH 8.5). K values at each membrane potential were obtained from fits to data from individual oocytes normalized to the Imax value obtained for that cell 50and are presented as means ± S.E. of 5–7 oocytes. F, the effect of membrane potential on maximum current (Imax) was determined at holding potentials of –10, –30, –50, and –70 mV in the presence of 100 mM external Na+ (pH 8.5) and a saturating concentration of uridine (100 µM). Each value is the mean ± S.E. of 3–4 oocytes from the same batch of cells used on the same day. No currents were observed in control water-injected oocytes. Note: to more accurately determine Km and K50 values (panels A–E), kinetic experiments at low membrane potentials were performed on preselected oocytes with maximal currents 40 nA. The effect of membrane potential on Imax (panel F) was measured independently in a separate experiment.

View larger version (32K): [in this window] [in a new window]   FIG. 7. Kinetic properties of Na+- and H+-coupled hCNT3. Initial rates of [14C]uridine uptake (20 °C, 1-min flux) were measured in Na+-containing (100 mM NaCl) and choline-containing (100 mM ChCl) transport media at either pH 7.5 (A and B, respectively) or pH 5.5 (C and D, respectively) in oocytes injected either with water alone (open circles) or with water containing hCNT3 RNA transcripts (solid circles). All of the fluxes were performed on the same batch of oocytes used on the same day. Kinetic parameters derived from these data for the hCNT3-mediated component of transport (uptake in RNA transcript-injected oocytes minus uptake in water-injected oocytes) are presented in Table I.

Na⁺/Nucleoside and H⁺/Nucleoside Coupling Ratios—The Na⁺/uridine and H⁺/uridine stoichiometries of hCNT3 were directly determined by simultaneously measuring uridine-evoked currents and [¹⁴C]uridine uptake under voltage clamp conditions, as described previously for SGLT1 (41), the rat kidney dicarboxylate transporter SDCT1 (42) and, most recently, for hCNT1 (32), hfCNT (30), and CaCNT (28). Each data point in Fig. 9 (A–F) represents a single oocyte, and the Na⁺/nucleoside or H⁺/nucleoside coupling ratio is given by the slope of the linear fit of charge (picomoles) versus uptake (picomoles) (Table III).

Charge-to-Na⁺ Stoichiometry—The relationship between uridine-evoked charge influx (picomoles) and ²²Na⁺ uptake (picomoles) was measured in five oocytes clamped at –90 mV (Fig. 10). A linear fit of the data gave a regression line with a slope of 0.97, indicating that one net inward positive charge was transported for every Na⁺ ion cotransported with uridine into the cell (Table III).

View larger version (16K): [in this window] [in a new window]   FIG. 8. Na+ and H+ activation of hCNT3. A, Na+ activation curve in a single representative hCNT3-producing oocyte measured in transport medium containing 0–100 mM NaCl (pH 8.5) at a uridine concentration of 5 µM and a membrane potential of –50 mV. Currents were normalized to the fitted Imax (±S.E.) of 53 ± 3 nA. The inset in A is a Hill plot of the data. The K50 was 7.0 ± 0.6 mM, and the Hill coefficient was 1.6 ± 0.2. No currents were observed in control water-injected oocytes. Results from mean Na+-activation experiments performed at different uridine concentrations are presented in Table II. B, H+-activation curve in a single representative hCNT3-producing oocyte measured in Na+-free transport medium (100 mM ChCl) at pH 8.5–4.5 and a membrane potential of –50 mV. Currents were normalized to the fitted Imax of 120 ± 11 nA. The inset in B is a Hill plot for the data. The K50 was 470 ± 99 nM, and the Hill coefficient was 0.68 ± 0.06. No currents were observed in control water-injected oocytes. Mean H+-activation data from six individual oocytes are summarized in Table II.

View larger version (22K): [in this window] [in a new window]   FIG. 9. Stoichiometries of Na+/uridine and H+/uridine cotransport by recombinant hCNT3. Charge to [14C]uridine uptake ratio plots were generated with 9 to 10 different hCNT3-producing oocytes in Na+-containing transport media (100 mM NaCl, pH 8.5) at membrane holding potentials Vh =–30 mV (A), Vh =–50 mV (B), and Vh =–90 mV (C). Similar conditions were used for D–F (n = 9) except that Na+ in the transport buffer was replaced by choline (100 mM ChCl), and the medium was acidified to pH 5.5. Integration of the uridine-evoked current was used to calculate the net cation influx (charge) and was correlated to the net [14C]uridine influx (flux). Linear regression analysis of the data for each plot is indicated by the solid line. The dashed line indicates a theoretical 2:1 charge:uptake ratio in A–C (presence of Na+) and a 1:1 charge:uptake ratio in D–F (presence of H+). Linear fits passed through the origin. Stoichoimetries (±S.E.) obtained from these data and from corresponding experiments performed in Na+-containing transport media (100 mM NaCl) at pH 5.5 (i.e. in the presence of both Na+ and H+) are summarized in Table III.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Unlike hCNT1 and hCNT2, which are largely Na⁺-specific, hCNT3 exhibited Na⁺, H⁺, and Li⁺ dependences, all three cations supporting electrogenic uridine influx. In this regard, hCNT3 resembles the mammalian concentrative glucose transporters SGLT1 and SGLT3, the bacterial MelB melibiose transporter, and the mammalian Na⁺/dicarboxylate cotransporter SDCT1/NaDC-1, all of which can also utilize Na⁺, H⁺, or Li⁺ to drive cellular accumulation of substrate (42–46). Consistent with an intracellular oocyte pH of 7.3–7.6 (47), nucleoside-evoked H⁺ currents were minimal at external pH values of 7.5 or higher. Use of CCCP to dissipate the imposed H⁺ electrochemical gradient across the cell membrane (48, 49) decreased the uridine-evoked current in acidified Na⁺-free medium by 60%, confirming that hCNT3 is coupled to the proton-motive force. H⁺ and Li⁺ coupling within the CNT3/hfCNT subfamily is unique to h/mCNT3 and is not shared by hfCNT (30). In contrast, some other CNTs function exclusively as H⁺/nucleoside symporters, including NupC from E. coli, CeCNT3 from C. elegans, and CaCNT from C. albicans (26–28).

Na⁺/nucleoside and H⁺/nucleoside symport by hCNT3 exhibited markedly different selectivity characteristics for physiological nucleosides and therapeutic nucleoside drugs, suggesting that Na⁺ and H⁺ binding induce cation-specific conformational changes in the hCNT3 substrate-binding pocket and/or translocation channel. For H⁺-coupled hCNT3, this was reflected in markedly reduced transport activity for thymidine, cytidine, adenosine, and inosine, and inability to transport guanosine, AZT, and ddC. In a possibly related phenomenon, functional studies with microglia have shown that an inwardly directed H⁺ gradient can inhibit AZT uptake (50). Microglia have cib-type activity as a major component of their nucleoside transport machinery (51).

Consistent with results of recent studies of hCNT1 (32), the proton/myo-inositol cotransporter (38), and SGLT1 (39), hCNT3 kinetic experiments revealed an ordered binding mechanism. Na⁺ removal increased the transporter's K_m for uridine by more than 30-fold, this being accompanied by a smaller (1.6-fold) increase in V_max. Limiting the concentration of Na⁺ can therefore be overcome by increasing the concentration of uridine to reach similar maximal rates of transport (37–40). In contrast, limiting the concentration of uridine reduced both the apparent affinity of hCNT3 for Na⁺ and the maximal current. Na⁺ therefore binds to hCNT3 first, followed by nucleoside. Like SGLT1 (44), the apparent affinity of hCNT3 for H⁺ was four orders of magnitude higher than for Na⁺. H⁺ and Na⁺ binding to SGLT1 also lead to cation-specific conformational changes, which, like hCNT3, were reflected in a decrease in sugar-binding affinity and transport efficiency of the H⁺-coupled transporter (45). In the case of hCNT3, substitution of H⁺ for Na⁺ resulted in a 6-fold change in uridine apparent affinity, and a decrease of 70% in V_max:K_m ratio (Table I). Unlike hCNT3, however, H⁺-coupled SGLT1 exhibited only modest changes in sugar specificity compared with the Na⁺-coupled transporter (45). Similar to SGLT1, hCNT3-mediated Na⁺/nucleoside and H⁺/nucleoside symport were voltage-dependent (35, 36). The apparent affinity of hCNT3 for uridine was voltage-insensitive at high external Na⁺ concentrations, but voltage-dependent when the concentration of Na⁺ was reduced, suggesting that the voltage dependence of the transporter's apparent affinity for uridine may be due to the voltage dependence of Na⁺ binding (35, 36). This was supported by experiments showing a similar voltage dependence of the apparent affinity of hCNT3 for Na⁺. As in the case of SGLT1, these results are indicative of the presence of an ion well effect (35, 36), with pre-steady-state electrophysiological studies suggesting that Na⁺ binds to hCNT3 40% within the membrane electric field (52). Na⁺/nucleoside and H⁺/nucleoside I-V curves and the effect of membrane potential on I_max values suggest that membrane potential also influences carrier translocation. This is consistent with the fact that the driving force for an electrogenic transporter is dependent on not only the gradients of substrate and cotransported ion, but also on the membrane potential.

View larger version (16K): [in this window] [in a new window]   FIG. 10. Charge-to-Na+ stoichiometry of hCNT3. The charge to 22Na+ uptake plot was generated from five different hCNT3-producing oocytes at a membrane potential of –90 mV. The sodium and uridine concentrations were 1 mM (pH 8.5) and 200 µM, respectively. Linear regression analysis of the data is indicated by the solid line (see Table III for the fitted slope). The dashed line indicates a theoretical charge: uptake ratio of 1:1. The linear fit passed through the origin.

View larger version (22K): [in this window] [in a new window]   FIG. 11. Transport properties of chimera hCNT3/1. A, topographical model of hCNT3 and hCNT1. Potential membrane-spanning -helices are numbered, and putative glycosylation sites in predicted extracellular domains in hCNT3 and hCNT1 are indicated by solid and open stars, respectively. Residues identical in the two proteins are shown as solid circles. Residues corresponding to insertions in the sequence of hCNT3 or hCNT1 are indicated by circles containing "+" and "–" signs, respectively. The arrow represents the splice site used for construction of the chimera. B, uptake of radiolabeled nucleosides by chimera hCNT3/1. Nucleoside influx (20 µM, 20 °C, 1-min flux) was measured in transport medium containing 100 mM NaCl at pH 7.5. Mediated transport was calculated as uptake in RNA transcript-injected oocytes minus uptake in control water-injected oocytes. C, radiolabeled uridine influx (20 µM, 20 °C, and 1-min flux) by hCNT1, hCNT3, and hCNT3/1 was measured in transport medium containing 100 mM NaCl at pH 7.5 (black bars) or in Na+-free transport medium (100 mM ChCl) at both pH 5.5 (gray bars) and 7.5 (open bars). Mediated transport was calculated as uptake in RNA-injected oocytes minus uptake in control water-injected oocytes. D, influx of [14C]uridine (20 µM, 20 °C, and 1-min flux) measured as a function of Na+ concentration at pH 7.5 using choline as Na+ substitute in oocytes injected with water alone (open circles) or with water containing hCNT3 RNA transcripts (solid circles). The inset in B is a Hill plot of the mediated data (Hill coefficient (n) and Na+ apparent affinity (K50) presented in the text). Values in B–D are means ± S.E. of 10–12 oocytes. Each experiment in B–D was performed on cells from single batches of oocytes used on the same day.

In marked contrast to SGLT1, where both Na⁺ and H⁺ have the same 2:1 cation/sugar stoichiometries (44, 59, 62), charge/uptake analyses revealed an hCNT3 Na⁺/nucleoside coupling ratio of 2:1 versus 1:1 for H⁺. Unlike Na⁺, the H⁺ coupling ratio was membrane potential-independent. Charge/uptake experiments with both Na⁺ and H⁺ present together (Na⁺-containing transport medium at pH 5.5) revealed features intermediate between Na⁺ or H⁺ alone (2:1 coupling ratio and voltage-independent), suggesting that both cations contributed to the driving force under these conditions. The 2:1 charge/uptake coupling ratio under these conditions implies that one of the two Na⁺ binding sites is shared by H⁺. Because proton-activation experiments gave a Hill coefficient consistent with a 1:1 H⁺/nucleoside coupling ratio, it is unlikely that there exists a second (recycled) H⁺ bound to hCNT3. The hCNT3 cation binding site shared by Na⁺ and H⁺ is likely to be the same as that responsible for single-site cation coupling in CNTs that are either exclusively H⁺-dependent (CaCNT, NupC) (26, 28) or Na⁺-dependent (hCNT1/2) (7–9, 32, 53).

We interpret our results for cation coupling of hCNT3 in terms of the conformational equilibrium model of secondary active transport developed by Krupka (63, 64). This modified ordered binding model of secondary active transport alleviates the stringent sequential carrier states of earlier models and instead allows for flexible cation interactions such as those observed for Na⁺ and H⁺ coupling of hCNT3. Because the transporter can accept two different solutes, cation (A) and nucleoside (S), it is proposed to exist in two inwardly facing or outwardly facing conformational states: one that binds cation only (T_i' and T_o') and one that binds both cation and nucleoside (T_i' and T_o'). Normally, the equilibrium between the two outwardly facing carrier states overwhelmingly favors the T_o' form and requires the addition of cation (two Na⁺ ions or one H⁺ in the case of hCNT3) to "unlock" or open the nucleoside binding site (T_o'A T_o'A), thereby promoting active transport. Both T_o'S and T_o'AS are considered mobile. The finding that binding of an H⁺ to one of the two Na⁺-binding sites is sufficient to activate nucleoside transport presents an experimental paradigm to enable mutageneic dissection of amino acid residues contributing to each of the sites. Our 50:50 hCNT3/1 chimera demonstrated that the structural determinants of cation/nucleoside stoichiometry and H⁺ dependence reside in the C-terminal half of the protein. Hill-type analysis of Na⁺/nucleoside coupling in a corresponding 50:50 chimera between hfCNT and hCNT1 yielded similar results (30). The present finding that determinants of hCNT1 versus hCNT3 nucleoside selectivity also reside in the C-terminal half of the protein is consistent with previous mutagenesis experiments that identified residues in TMs 7 and 8 of hCNT1 that, when sequentially mutated to the corresponding residues in hCNT2, progressively changed the selectivity of the transporter from cit to cib to cif (29).

In conclusion, hCNT3 exhibited unique cation interactions with Na⁺, H⁺, and Li⁺ that are not shared by other members of the CNT protein family. Both indirect and direct methods indicated 2:1 and 1:1 cation/nucleoside stoichiometries for Na⁺ and H⁺, respectively, and Na⁺- and H⁺-coupled hCNT3 exhibited markedly different selectivities for nucleoside and nucleoside drug transport. Location of hCNT3-specific cation interactions to the C-terminal half of the protein sets the stage for site-directed mutagenesis experiments to identify the residues involved. The ability of hCNT3 to couple nucleoside and nucleoside drug accumulation to H⁺ as well as Na⁺ cotransport may have physiological and pharmacological relevance in the duodenum and proximal jejunum where the pH of luminal contents can be relatively acidic. As well, there is a reported acidic microenvironment present on the surface of the intestinal epithelium (58).

	FOOTNOTES

 
^* This work was supported in part by the National Cancer Institute of Canada, with funds from the Canadian Cancer Society, the Alberta Cancer Board, the Heart and Stroke Foundation, Canada, and the Medical Research Council, UK. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors contributed equally to this work.

^¶ Funded by Studentships from the Alberta Heritage Foundation for Medical Research.

A Canada Research Chair in Oncology.

^¶¶ A Heritage Scientist of the Alberta Heritage Foundation for Medical Research. To whom correspondence should be addressed: Dept. of Physiology, 7-55 Medical Sciences Bldg., University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Tel.: 780-492-5895; Fax: 780-492-7566; E-mail: james.young{at}ualberta.ca.

¹ The abbreviations used are: NT, nucleoside transporter; CNT, concentrative nucleoside transporter; ENT, equilibrative nucleoside transporter; AZT, 3'-az